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Abstract
Peroxynitrite is a potent nitrating and oxidizing agent that exerts differential effects on platelets. In the present study we investigated the influence of peroxynitrite on vasodilator-stimulated phosphoprotein (VASP), a protein that plays a key role in inhibition of platelet adhesion and spreading. In platelets, VASP is a substrate for protein kinase A (PKA), PKC and PKG and phosphorylation by these kinases is thought to block VASP-mediated actin cytoskeletal rearrangement. In the present study, we demonstrate that peroxynitrite phosphorylates VASP by a PKC-dependent mechanism. Peroxynitrite (0–100 µM) induced a concentration and time-dependent increase in phosphorylation of VASP at serine157 (Ser157) and Ser239. Inhibition of soluble guanylyl cyclase (sGC) did not significantly reduce peroxynitrite-mediated phosphorylation, indicating a cGMP-independent pathway for VASP phosphorylation. In contrast nitric oxide-mediated VASP phosphorylation was abolished under conditions of sGC inhibition. Further exploration of the mechanisms underlying VASP phosphorylation indicated a requirement for Ca2+ mobilization, but was independent of protein kinase A, Src kinases and protein nitration. Consistent with previous reports phorbol 12-myristate 13-acetate (PMA; 300 nM) induced phosphorylation of VASP at Ser157, but not Ser239, which was blocked by general protein kinase C (PKC) inhibitors, Ro31-8220 and Bisindolylmaleimide I (BIM-1), and Gö6976, an inhibitor of conventional PKC isoforms. Interestingly, treatment of platelets with these PKC inhibitors significantly reduced peroxynitrite-mediated phosphorylation of both sites, indicating that phosphorylation occurred through PKC-dependent mechanism. Consistent with these findings peroxynitrite caused a small increase in PKC activity as evidenced by increased phosphorylation of PKC substrates. Together these data indicate that peroxynitrite may inhibit platelet function by inducing the phosphorylation of VASP through a mechanism that requires the activation of PKC.
| Abbreviations | ||
| BIM-1 | = | Bisindolylmaleimide I |
| cGMP | = | cyclic guanosine monophosphate |
| GSNO | = | S-Nitrosoglutathione |
| H89 | = | N-[2-(p-Bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide |
| HS | = | human serum |
| NO | = | Nitric oxide |
| ODQ | = | 1H-[1, 2, 4]Oxadiazolo[4,3-a]quinoxalin-1-one |
| sGC | = | soluble guanylyl cyclase |
| PKA | = | Protein kinase A |
| PKC | = | Protein kinase C |
| PKG | = | Protein kinase G |
| WP | = | Washed platelets |
| VASP | = | vasodilator stimulated phosphoprotein |
| Abbreviations | ||
| BIM-1 | = | Bisindolylmaleimide I |
| cGMP | = | cyclic guanosine monophosphate |
| GSNO | = | S-Nitrosoglutathione |
| H89 | = | N-[2-(p-Bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide |
| HS | = | human serum |
| NO | = | Nitric oxide |
| ODQ | = | 1H-[1, 2, 4]Oxadiazolo[4,3-a]quinoxalin-1-one |
| sGC | = | soluble guanylyl cyclase |
| PKA | = | Protein kinase A |
| PKC | = | Protein kinase C |
| PKG | = | Protein kinase G |
| WP | = | Washed platelets |
| VASP | = | vasodilator stimulated phosphoprotein |